cost and benefits of the morphological defences in larvae ... · larvae of scymnus nubilus mulsant...
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PAULO ALEXANDRE BOTELHO PACHECO
Cost and benefits of the morphological defences in
larvae of Scymnus nubilus Mulsant
(Coleoptera: Coccinellidae)
DEPARTAMENTO DE BIOLOGIA
UNIVERSIDADE DOS AÇORES
PONTA DELGADA
2011
PAULO ALEXANDRE BOTELHO PACHECO
Cost and benefits of the morphological defences in
larvae of Scymnus nubilus Mulsant
(Coleoptera: Coccinellidae)
Dissertação para a obtenção de Grau de Mestre em Biotecnologia em Controlo Biológico
Orientador: Doutor António Onofre Soares
Co-orientadores: Doutora Isabel Borges & Doutor Eric Lucas
DEPARTAMENTO DE BIOLOGIA
UNIVERSIDADE DOS AÇORES
PONTA DELGADA
2011
ACKNOWLEDGEMENTS
Agradeço:
Ao orientador, o Doutor António Onofre Soares cujo rigor, exigência, ajuda disponibilizada e
acompanhamento (mesmo quando me atrasava) permitiram-me terminar este trabalho;
À co-orientadora Doutora Isabel Borges, pela sua disponibilidade, paciência, esclarecimentos
prestados e revisão crítica do meu trabalho;
Ao Doutor Eric Lucas, pela análise e crítica ao trabalho desenvolvido;
Ao Doutor Luís Silva pela identificação das espécies de plantas recolhidas no campo;
Ao Doutor Juan Nafria pela ajuda na identificação dos afídeos;
Ao Roberto Resendes, pela sua assistência na produção dos insectos em laboratório;
Ao Mário Teixeira, por escutar as minhas divagações epistemológicas entre os intervalos do
trabalho laboratorial;
E, finalmente, à Maria João Melo, um agradecimento especial pelo seu apoio e por me ter
relembrado constantemente que o meu objectivo principal era a realização deste trabalho.
INDEX
ACKNOWLEDGEMENTS ............................................................................................................... III
ABSTRACT………………………………………………………………………………………....V
RESUMO ........................................................................................................................................... VI
1. INTRODUCTION ........................................................................................................................... 1
2. METHODS ...................................................................................................................................... 3
2.1. Field work ................................................................................................................................. 3
2.2. Insect rearing………………………………………………………….....................................4
2.3. Development time, adult body weight and ovarioles number……………………………......4
2.4. Larval alimentary physiology ………………………………………………………..............5
2.5. Intraguild Predation…………………………………………………………………………..6
2.6. Statistical analysis…………………………………………………………………………….7
3. RESULTS……………………………………………………………………………………….....8
3.1. Field work……………………………………………………………………………….…....8
3.2. Development time, adult body weight and ovarioles number………………………………..9
3.3. Larval alimentary physiology……………………………………………………………….12
3.4. Intraguild predation………………………………………………………………………….14
4. DISCUSSION…………………………………………………………………………………….14
5. REFERENCES………………………………………………………………………………..….16
Abstract
Among insects different defence mechanisms evolved against predation and parasitism.
Defences can be chemical, behavioural and/or morphological. The larvae of the small
aphidophagous coccinellid Scymnus nubilus are covered by a wax layer that might act as a
defensive mechanism against ants and other predators. However, the degree of protection conferred
by waxes in Scymnus larvae is not very well known.
The objective of the study was to evaluate some of the costs and benefits of wax production in
S. nubilus. According to life history evolution theory, increased performances in one trait entail a
decrease in another trait. If wax production is metabolically costly to larvae, we expect some costs
on developmental time, growth or future reproductive potential. Because potential trade-offs are
only evident when the organism are under stressful conditions, we evaluated the consequences of
wax production to developmental time, alimentary physiology and potential reproductive effort in
larvae, either wax and waxless (artificially removed).
From a physiological point of view we expect waxless larvae will display higher prey
consumption in order to meet metabolic requirements for growth and wax production. To balance
these costs and energetic allocation we propose that waxes might act as a defensive mechanism
against an intraguild predator, the lacewing Chrysoperla agilis.
Our results showed that there were differences between the biological parameters of wax and
waxless larvae, specifically, in the larval growth. Contrary to our prediction, prey consumption of
wax and waxless did not vary significantly although relative weight gain was higher in wax larvae.
This indicates that in order to produce waxes, resources need to be re-allocated and growth is
reduced. Waxes reduced intraguild predation as predicted.
Key-words: Scymnus nubilus, morphological defence, cost-benefits of waxes, alimentary
physiology, trade-offs, intraguild predation
Resumo
Diferentes mecanismos de defesa contra a predração e parasitismo evoluíram entre os
insectos. Estes podem ser químicos, comportamentais ou estruturais. As larvas do pequeno
coccinelídeo Scymnus nubilus estão cobertas por uma camada de ceras que podem actuar como um
mecanismo defensivo contra formigas e outros predadores. No entanto, o grau de protecção
conferido pelas ceras às larvas de Scymnus não é bem conhecido.
O objectivo deste estudo foi avaliar alguns dos custos e benefícios da produção de ceras nos S.
nubilus. De acordo com a teoria da evolução das histórias de vida, um maior desempenho de um
traço específico implica um decréscimo noutro traço. Se a produção das ceras é metabolicamente
dispendiosa para as larvas, esperamos alguns custos no tempo de desenvolvimento, crescimento e
futuro potencial reprodutor. Devido ao facto de potenciais trade-offs serem apenas evidentes quando
um organismo está sobre condições de stress, avaliamos as consequências da produção de ceras no
tempo de desenvolvimento, fisiologia alimentar e potencial reprodutor nas larvas com e sem ceras
(removidas artificialmente). Do ponto de vista fisiológico, espera-se que a remoção das ceras das
larvas induzirá um consumo maior de presas de modo a que se cumpram os requerimentos
metabólicos necessários para o crescimento e produção de ceras. A equilibrar estes custos e a
alocação energética, propomos que as ceras actuarão como um sistema defensivo contra a C. agilis
um predador intraguilde.
Os resultados demonstram que existiram diferenças entre os parâmetros biológicos das larvas
com e sem ceras, mais especificamente no crescimento larval. Ao contrário do previsto, o consumo
de presas das larvas com e sem ceras não variou, embora o peso ganho tenha sido maior nas
primeiras. Isto é uma indicação que para produzir as ceras há necessidade de distribuir os recursos e
o crescimento é reduzido. As ceras reduziram a predação intraguilde, tal como esperado.
Palavras-chave: Scymnus nubilus, defesa morfológica, Custos-benefícios das ceras, fisiologia
alimentar, trade-offs, predação intraguilde
1
Introduction
Among insects different defence mechanisms evolved against predation and parasitism.
Defences can be chemical, behavioural and/or morphological. The majority of coccinelids are
protected by chemical defences (Sloggett et al., 2011). Reflex bleeding is an example of a chemical
defence in which coccinelid larvae and adults emit droplets of haemolymph that are distasteful and
toxic (Hemptinne & Dixon, 2000). In ant-tending aphid colonies, a common behaviour in order to
avoid ant attacks is to cover the larvae body with dead aphid such as in the case of Aphidoletes
aphidimyza Rondani, or the green lacewing Mallada desjardinsi Navas (Lucas, 2005; Hayashi &
Nomura, 2011). At the morphological level, the production of a wax cover, such as the ones found
on larvae of Scymnus sp., act as a protection against predation and ant aggression (Völkl & Vohland,
1996).
Scymnus nubilus Mulsant is a small aphidophagous ladybird frequent in the Mediterranean
region and also present in the Azores islands (Soares et al., 2003; Soares et al., 2005; Soares, 2010).
Compared to large-sized ladybirds Scymnus spp. may have weaker competitive abilities.
Nevertheless, by surviving with low prey densities Scymnus are able to exploit aphid colonies at an
earlier stage than larger ladybirds (Agarwala & Yasuda, 2001). Larvae of this species are covered by
a thick wax layer produced by dorsal epidermal cells (Agarwala & Yasuda, 2001). This
morphological defensive mechanism is found in several insect orders and has been associated with
protection against the attack of predators or parasitoids (Eisner, 1994; Takabayashi & Takahashi,
1993; Völk & Vohland, 1996). This morphological defense mechanism balances their apparent
absence of chemical defenses. Other functions have been attributed to the waxes such as protection
against UV radiation and reduction of water transpiration (Agarwala & Yasuda, 2001). Their sticky
nature not only makes the larvae difficult to bite into but also serves as an entangling agent. In the
case of several Scymnus species these have been observed physically clogging the mouthparts of
2
aggressive ants (Agarwala & Yasuda, 2001; Schwartzberg et al., 2010; Völk & Vohland, 1996).
There are evidences that the larval wax also acts as mimetic mechanism. This has been found in
some species that feed on mealy aphids attended by ants. In this case the larvae are not recognized
as a threat (Majerus et al., 2007). Recently Schwartzberg et al. (2010) tested if the wax structures of
S. louisianae J. Chapin acted as a defense mechanism against Lasius neoniger Emery. The results
showed that the wax attenuated ant aggression towards the ladybeetle when compared to denuded
larvae. Evolutionary, the success of Coccinellidae can be associated to the exploration of ant-tended
insects as a food source which favored the development of defenses mechanisms such as larval
waxes (Seago, et al., 2011).
S. nubilus does not occur isolated in their natural habitat. They belong to aphidophagous
guilds and thus is subjected to an array of biotic interactions. Intraguild predation (IGP) is a type of
interaction that occurs between species that share a common resource. It is an extreme form of
competition that can have various ecological effects such as the alteration of the distribution,
abundance and evolution of the species involved (Lucas et al., 1998; Félix & Soares, 2004). Besides
the protection given by the waxes against ant attacks, any additional protection against IGpredators
would be beneficial to the ladybeetle. Not much is known about the defensive potential of the
Scymnus spp. waxes against several IGpredators. Völk & Vohland (1996) studied this subject
regarding the carabid beetle Platynus dorsalis Pontoppidan and Agarwala & Yasuda (2001) with the
syrphid larvae Eupeodes freguens Matsumura.
The objective of this study was to evaluate the costs and benefits of wax production in S.
nubilus. It is expected that the production of waxes will be metabolically costly. According to life
history evolution theory, increased performances in one trait entail a decrease in another trait.
Considering wax production is, in metabolic terms, costly (Eisner, 1994) we expect some
detrimental effects on developmental time, size or reproductive potential. Under a physiological
point of view we expect that removing the waxes from larvae will induce higher prey consumption
3
in order to meet metabolic requirements for growth and wax production. We also predict that wax
cover confer protection against IGpredation.
2. Material & Methods
2.1. Field work
In a previous study the occurrence of S. nubilus waxless larvae was observed in the laboratory
(Borges, pers. comm.). It was estimated that 10% of larvae did not developed waxes during the
larval development. This is not reported in the literature.
In order to seek for the co-occurrence of wax and waxless Scymnus spp. larvae, adults and
potential IGpredators, several aphid colonies were followed during a field work undertaken on the
island of São Miguel, Azores (Portugal) in two different habitats; (i) corn fields and (ii) a coastal
area (Santana). In 2011 corn fields were surveyed from June to November and the coastal area from
June to August. In 2010 an exploratory field work was carried out to search for the co-occurrence of
wax and waxless larvae. Considering the homogeneity of the corn field habitats, aphid colonies
were randomly selected and checked once a week. For each plant there was a sampling time of three
minutes. For the two years, the total corn plants surveyed was in a total of 44. In the coastal area
(Santana) due to the heterogeneity of the habitat, the sampling method was adapted from Borges et
al. (2011). Twenty sampling plots were disposed along two linear transects. Each sampling plot was
a circular area of 5 m in diameter and separated from each other by 10 m. In each plot aphid
colonies were selected and marked on different host plants. From these, a sample of aphids was
collected and preserved on 70% ethanol. These samples were sent for identification to a specialist.
Twice a week the number and size of aphid colonies as well as the presence of Scymnus spp. or
potential IGpredators was registered.
4
2.2. Insect rearing
Larvae and adults of S. nubilus collected from corn fields were kept and reared in 2 L plastic
cages at approximately 20±1ºC, 75±5% RH and a light regime of 16L:8D. A mixed diet of Myzus
persicae (Sulzer) and Aphis fabae Scopoli reared on broad bean plants (Vicia faba L. Major) was
provided ad libitum. Every two days the infested plants were replaced by new ones. Once a week,
larvae and adults were transferred into new rearing cages. The eggs were separated into another 2 L
plastic cage until larval eclosion.
Chrysoperla agilis Henry, Brooks, Duelli & Johnson (Neuroptera: Chrysopidae) is a common
lacewing species found in the Azores (Borges et al., 2010) and a potential IGpredator for S. nubilus.
They were reared in net cages at 20º±1ºC, 75±5% RH and a light regime of 16L:8D. Adults were
fed with a mixture of honey, yeast and pollen grains dissolved in water. After the emergence of
larvae, aphids were provided in broad bean plants until adult emergence. Once a week, old and new
adults were transferred into a new cage.
2.3. Development time, adult body weight and ovarioles number
This experiment had two treatments: wax and waxless. During the first, larval wax was never
removed whereas on the second one the waxes were removed artificially with a brush once a day
until pupation.
To determine the existence of potential trade-offs in wax and waxless individuals the
development time, adult body weight and ovarioles number of S. nubilus was determined. Twenty-
five couples were separated into 5 cm diameter x 2 cm height plastic boxes (5 couples per box).
They were allowed to reproduce for 24 hours in order to obtain fresh eggs. The adults were then
5
removed and the eggs were observed twice a day until eclosion of larvae. New eclosed larvae were
weighed and individually kept in 3 cm diameter x 1 cm height plastic boxes. Larval stage was
checked twice a day at 9am and 4pm for the presence of exuviae. The development time from egg
to adult was determined.
The adults were weighed 12h after emergence and sex was determined according to the
coloration of the head: yellowish for males and predominantly dark brown for females (Raimundo
& Alves, 1986). After 5 days, S. nubilus females were dissected in order to determine the number of
ovarioles. All weighings were made in a Sartorius ultramicrobalance SE 2.
Individual fitness (r) was adapted from Sadeghi & Gilbert (1999, 2000):
R=[ln(m.V)]/D
where ln = natural logarithm, m = survival (1 or 0), V = potential fecundity and D =
development time. Fitness was only calculated for surviving females where D is the value of their
development time. Since this is the case, the value given for the variable m was always 1. The value
of potential fecundity was given by the ovarioles number of each female.
2.4. Larval alimentary physiology
Larval alimentary physiology tests were performed with 12 hours old 4th instar larvae
obtained as described previously. Thirty larvae (15 wax and 15 waxless) were isolated and subjected
to 12 hours of starvation before conducting the tests. For the wax treatment, the waxes of the larvae
were not removed during their development. For the waxless treatment the waxes were removed
daily since larval eclosion. Larvae were provided with 40 M. persicae adults for 24 hours. Both,
larvae and aphid biomass were determined, before and after the test. In order to determine the
6
weight loss due to dehydration, 40 M. persicae were confined in 3 cm plastic boxes 3cm diameter x
1 cm during 24h. They were weighed before and after the referred period.
Biomass consumption (BC) and weight gain (WG), expressed in absolute and relative terms,
were calculated. Conversion efficiency (CE%) was also calculated (adapted from Borges, 2008):
where PWi is the prey initial weight, PWf the final prey weight and PWd the aphid weight
loss due to dehydration. LWi and LWf are the larval initial and final weights respectively.
2.5. Intraguild Predation
To test if the presence of waxes provided a defensive morphological structure against
IGpredation two treatments were made: wax and waxless. Waxes were not removed on the wax
treatment and for waxless these were only removed before conducting the test.
The developmental stages of the predators were selected according to weight so this would
not be a factor conditioning the intraguild predation direction. For S. nubilus (0.91±0.09 mg) and C.
agilis (0.93±0.07 mg) (t-test: t = 0.186, p>0.05). The selected instar for S. nubilus was the fourth
and the second for C. agilis.
In order to obtain the larval stages, eggs of S. nubilus and C. agilis and were separated daily
7
from rearing cages and transferred to 3 cm x 1 cm plastic boxes. Their development was checked
twice a day. After molting into the selected instars, larvae were fed for 12h followed by a period of
24h of starvation to increase their hunger.
Before the experiment, one S. nubilus and one C. agilis larvae were weighed and placed in a
petri dish for 24h. After this period the predators were examined under a stereoscope to determine
survival of the predators (n=15).
For each treatment, a test control to estimated survival rate was performed. Fifteen larvae of
each species were isolated in a petri dish for 24h.
IGP levels were estimated from the rates of predation for S. nubilus (RPsn) and C. agilis
(RPca), which were calculated as follows (Félix & Soares, 2004):
RPsn = (P(ca,sn) SRca / N) 100
RPca = (P(sn,ca) SRsn / N) 100
where “P(ca,sn)” = number of individuals of C. agilis killed, “P(sn,ca)” number of individuals of S.
nubilus killed, “SRca” survival rate of C. agilis in control, “SRsn” survival rate of S. nubilus in
control and “N” number of replicates. Survival rates were calculated from the control tests.
The symmetry index of Lucas et al. (1998) was used. This index expresses the number of
replicates in which a given predator was eaten over the total number of replicates where IGP
ocurred.
2.6. Statistical analysis
For the trade-offs experiment, the t-test was used to compare the time of development, adult
weight, number of ovarioles and fitness of wax and waxless larvae. The t-test was also used to
compare wax and waxless prey consumption (either in absolute and relative terms), weight gain
8
(either in absolute and relative terms) and conversion efficiency. Data normality and variance
homogeneity were assessed by the Kolmogorov-Smirnov and Levene´s tests, respectively. All data
was analysed with SPSS (2010) statistical package version 19.
Concerning the IGP test, the symmetry indices for each combination were compared with the
theoretical index of 50% corresponding to a symmetrical interaction, using a Chi-square test (SPSS,
2010). The strength of the IGP between the two species for a given combination was assessed using
the χ2 and was considered (i) symmetrical, when the χ2 value was not significant, which indicated
that the rate of predation of the two species was similar, (ii) asymmetrical, when the χ2 value was
significant and (iii) not significant asymmetrical, when the χ2 value was not significant but the rate
of predation of the two species differed.
3. Results
3.1. Field work
In 2010, in corn field, 29 Scymnus spp. were observed from a total of 24 prey colonies. From
these, 75.9% were larvae and 24.1% adults. The only IGpredator observed was Aphidoletes sp (9
larvae). In 2011 the number of ladybirds was higher with 47 Scymnus, from 21 aphid colonies
(59.6% larvae and 40.4% adults). One hundred and twelve Aphidoletes sp. were observed
throughout the colonies and one lacewing larva on an aphid colony. Mummies of aphids were also
observed.
The vegetation cover of the coastal area is presented in Table 1. On the coastal area, a total of
53 Scymnus spp. were observed from 97 prey colonies. Only 11.3% of these were adults. The
highest number of Scymnus larvae was found on Arundo donax L. and Tropaeolum majus L.. Four
plant species did not have any ladybird larvae or co-occurring predators (Table 1). Aphidoletes sp.
9
was again the most abundant IGpredator with a total of 116 individuals versus two syrphids and one
coccinelid larvae. These last IGpredators were observed individually on a colony of Aphis
oenotherae and Aphis fabae, respectively (Table 1).
Naturally occurring waxless larvae were not observed during the field work.
Table 1- List of the plant species found in Santana and their corresponding aphids, number of S. nubilus larvae
observed and co-occuring predators.
3.2. Development time, adult body weight and ovarioles number
Male wax larvae developed significantly faster than the waxless larvae (t-test: t = -3.195,
p<0.005) but for females no significant difference was found (t-test: t = -0.407, p>0.05) (Figure 1).
Larvae Adults Ants Aphidoletes sp. Syrphid Coccinelid
Arundo donax L. Melanaphis donacis Passerini 13 0 + + - -
Chrysanthemum segetum L. Aphis sp. 1 1 + - - -
Daucus carota L. Aphis fabae Scopoli 5 1 + + - -
Galactites tomentosa Moench Brachycaudus cardui L. 0 0 + - - -
Malva pseudolavatera Webb & Berthel Aphis fabae Scopoli 3 2 + + - -
Mentha suaveolens Ehrh. Aphis ruborum Börner 0 0 + + - -
Oenothera biennis L. Aphis oenotherae Oestlund 2 1 + + + +
Opuntia stricta (Haw.) Haw. Aphis fabae Scopoli 0 0 + - - -
Parietaria judaica L. Aphis fabae Scopoli 7 0 + + - -
Rubus ulmifolius Schott Aphis ruborum Börner 1 0 + + - -
Rumex aquaticus L. Aphis fabae Scopoli 0 0 + - + -
Solanum mauritianum Scop. Aphis fabae Scopoli 0 1 + - - -
Sonchus asper (L.) Hill Uroleucon sonchi L. 3 0 + - - -
Tropaeolum majus L. Aphis fabae Scopoli 12 0 + + - -
Host Plant Aphid speciesScymnus sp. Main co-occurring predators and ants
10
Figure 1- Development time (days±SE) from egg to adult for males and females of wax and waxless S. nubilus (for
each sex different letters mean significant differences).
There was a significant difference between both treatments for the adult weight at emergence
(Figure 2). The male (t-test: t=8.036, p<0.001) and female (t-test: t=4.766, p<0.001) adult weights
were significantly higher when no waxes were removed during the development of the larvae.
There was no significant difference between treatments for the ovarioles number (t-test: t=-
1.326, p>0.05) and fitness (t-test: t = -0.877, p>0.05) (Figure 3).
a a b a
0
5
10
15
20
25
30
35
Males Females
Dev
elop
men
t ti
me
(days)
Wax
Waxless
11
Figure 2- Mean weight (mean±SE) of S. nubilus adults at emergence.
Figure 3- Mean number (mean±SE) of ovarioles for the wax and waxless S. nubilus female adults.
a
a
b
b
0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
Males Females
Wei
gh
t (m
g)
Wax
Waxless
a a
0
2
4
6
8
10
12
Wax Waxless
Nu
mb
er o
f ovari
ole
s
12
Figure 4- Individual fitness of wax and waxless S. nubilus larvae.
3.3. Larval alimentary physiology
There was no significant difference on aphid consumption during 24h between the wax and
waxless treatments in absolute terms (t-test: t=0.968, p>0.05) and relative terms (t-test: t=1.328,
p>0.05) (Figure 5 and 6).
Figure 5- S. nubilus larvae absolute consumption (mean±SE) of aphids during 24h.
a a
0,00
0,02
0,04
0,06
0,08
0,10
Wax Waxless
Ind
ivid
ual fi
tnes
s
a a
0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
4,0
4,5
Absolute consumption
Co
nsu
mp
tio
n (
mg
)
Wax
Waxless
13
Figure 6- S. nubilus larvae relative consumption (mean±SE) of aphids during 24h.
The absolute weight gain (t-test: t=2.025, p=0.052) (Figure 7) and the conversion efficiency
(t-test: t=1.114, p>0.05) (Figure 9) did not differ for waxy and waxless larvae. However if weight
gain was expressed relatively to body weight (Figure 8) a significant difference was detected (t-test:
t=2.488; p<0.05).
Figure 7- Absolute weight gain (mean±SE) (mg) for each of the treatments during 24 hours.
a
a
0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
4,0
4,5
Relative consumption
Co
nsu
mp
tio
n (
%)
Wax
Waxless
a
a
0,0
0,5
1,0
1,5
Absolute weight gain
Wei
gh
t gain
(m
g)
Wax
Waxless
14
Figure 8- Relative weight gain (mean±SE) (mg) for each of the treatments during 24 hours.
Figure 9- Feeding efficiency (mean±SE) of the larval treatments.
3.4. Intraguild predation
The weight of S. nubilus and C. agilis did not show statistical differences for the wax (t-test:
t=0.186, p>0.05) and waxless treatment (t-test: t=-1.267, p>0.05).
All waxless larvae were preyed by C. agilis whereas the rate of predation decreased to 79% in
a
b
0,0
0,5
1,0
1,5
Relative weight gain
Wei
gh
t gain
(%
) Wax
Waxless
a
a
0
10
20
30
40
50
Wax Waxless
Con
ver
sion
eff
icie
ncy
(%
)
15
the combination with the wax S. nubilus larvae. In one replicate of the wax treatment both larvae
survived. There was a significant asymmetrical IGP in the combination C. agilis versus waxless S.
nubilus (χ2=0.001, df=1, p<0.05). No significant asymmetry was observed for the other treatment
(χ2=0.037, df=1, p>0.05)-
Figure 6- Rate of predation (RP) of the fourth instar of S. nubilus and second instar of C. agilis for the two ladybird wax and waxless
treatments. * indicates significant asymmetrical IGP.
Discussion
Contrarily to our expectations, no natural occurring waxless larvae were found in the field
work. These expectations were due to the fact that previous observations made in the laboratory
conditions revealed that the some larvae of S. nubilus did not produce wax during their development
(Borges, pers. comm.). The absence of waxless larvae could be associated either to an expected
susceptibility to intraguild predation, as our results seem to indicate, or a low probability to find
them due to low frequency.
As hypothesized, the stress of removing the wax cover in larvae resulted in some differences
in the biological parameters and emphasized a trade-off between the investment on larvae wax
0
50
100
Wax Waxless
*
Rate
of
pre
dati
on
(%
)
C. agilis
S. nubilus
16
production and adult biomass, Indeed some trade-offs are only evident when the organism is under
stressful conditions (Stearns, 2004). The daily artificial removal of waxes in the larvae resulted in
smaller adults. Nevertheless females of the wax and waxless treatments showed the same rate of
development and reproductive investment in gonads. There was, therefore, a reduction of
investment in soma in order to maintain fitness. Sato et al. (2009) showed that Harmonia axyridis
Pallas larvae forced to reflex bleed originated lighter adults than adults developed from larvae
where no stimulation was given.
Contrary to what we predicted, waxless larvae did not increase prey consumption, in both
absolute and relative terms. Absolute weight gain was also not statistically different between
treatments. However the significance of the test is close to the 0.05 limit of significance. This result
is not completely unexpected because wax larvae were heavier than waxless ones. Once the initial
larval weight was factored out, statistical differences arose. Conversion efficiency was similar for
both treatments. This seems to indicate that there is a displacement of resource allocation caused by
the wax removal. While the consumption and conversion efficiency into biomass did not differ,
there is indication that there was an investment made in the wax production over growth, from the
waxless larvae. The organisms must allocate the food resources between metabolism, growth and
reproduction. If the energy acquired only covers metabolic costs then the larvae will disinvest in
body growth (Hodek & Honĕk, 1996). The results suggest that removing the waxes will entail a
large resource allocation to regeneration of the cottony cover, leaving less matter and energy to
devote to body growth. Fourth instar larvae have greater metabolic costs due to the process of
preparation for pupation (Hodek & Honĕk, 1996). Although the larval alimentary physiology was
not tested for all instars, it may be that the fourth instar is more vulnerable to the induced stress of
removing the waxes. Indeed, the initial body weight of the fourth instar larva from wax and waxless
treatments was similar (t-test: t=-0.537, p>0.05).
Concerning to IGP, as we expected, the larval wax cover seems to act as an intraguild defense
17
mechanism against C. agilis although to a small extent since the rate of predation of C. agilis was
79%. Nevertheless this is an indication of their defensive potential against an IGpredator such as the
lacewing that has greater aggressiveness (Lucas et al., 1998). Even though wax-producing
coccinellid predators seem to have evolved to exploit ant-tended soft body sternorrhynchan insects
as a food source (Seago et al., 2011), they may have a secondary function as a defensive mechanism
against other intraguild predators. Völkl & Vholand (1996) demonstrated that they effectively
enhanced Scymnus interruptus Goeze survival against the carabid beetle Platynus dorsalis
Pontopiddan. Agarwala & Yasuda (2001) found that Scymnus posticalis Sicard waxes were also
effective against the attack of syrphids although the protection was not absolute. Besides the small
size of S. nubilus that allows them to forage in areas of low prey density and the ability to prey on
ant-tending aphid colonies (Agarwala &Yasuda, 2001) at a time when bigger predators have not yet
been established (Schwartzberg et al., 2011), the waxes also confer some degree of protection
against IGpredators that may explore the same resources spatially and temporally.
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